Fig. 5: Identification of Tet-responsive Twister ribozymes by amplicon-seq.

a Secondary structure of the Tet-Twister library design. Blue boxes indicate the randomized motifs of the nine sub-libraries, connecting the Twister ribozyme and the Tet aptamer. The arrowhead indicates the cleavage site. GU/UG mutation at the indicated position renders the ribozyme inactive. Inset: Library construct frequency analysis by DNA sequencing. b Volcano plots displaying the significance (FDR) of changes in riboswitch abundance as a function of the change in expression (log₂ fold change) for the nine Tet-Twister sub-libraries. Selected hits are labeled with their sequence motif. c Selected hits from the Tet-Twister library screen, randomized sequence, mean counts per million (CPM), fold change (FC) and false-discovery rate (FDR) at 12.5, 25, and 50 µM Tet, respectively. The bar plots show the raw counts under untreated, 12.5, 25, and 50 µM Tet-stimulated conditions (four groups from left to right, n = 8 replicates each). d Functional hit validation in HeLa cells at increasing tetracycline doses [µM] (n = 3 replicates, mean ± s.d.); psi-Check: ribozyme-free control construct; Tw-act/inact: constitutively active/inactive ribozyme control. e Maximal fold changes and p-values measured upon stimulation vs. 0 µM Tet for all tested constructs. f Network analysis of motif similarity for the CG_3N3N library at 25 µM Tet vs. untreated and an FDR < 1%. Each line segment represents a single nucleotide change. The “T9” construct is circled in red. *p < 0.05, **p < 0.01, ***p < 0.001 (unpaired T-Test, two-tailed). Source data are provided as a Source Data file.